To date, production of proteins for pharmaceutical applications via gene recombination techniques has mainly involved the use of animal cells or E. coli cells as hosts. E. coli cells enable the production of target proteins at low cost; however, E. coli cells cannot undergo modification typified by sugar chain modification, and inactive proteins are produced in the inclusion bodies. This requires a process of solubilization, and thus, E. coli cells are not suitable for complicated procedures for protein production. In contrast, animal cells enable the production of target proteins as active proteins. Disadvantageously, however, use of animal cells would remarkably increase production costs in terms of equipment and material costs, due to time-consuming operations of breeding and culture of animal cells.
Among proteins, antibodies have been used as pharmaceutical products for a long time. Since they were derived from sources other than humans, antibodies against the administered antibodies were newly produced. Thus, such antibodies could not be administered more than once, and use thereof was restricted. In recent years, production of a humanized antibody in which an amino acid sequence other than the antigen-binding site has been substituted with a sequence derived from a human antibody became possible. Further, production of a human antibody-producing mouse into which a human antibody gene has been introduced became possible. Thus, application of antibodies as pharmaceutical products became extensive. At present, such antibodies are produced by cultured cells, such as CHO cells, into which genes encoding hybridomas or antibodies have been introduced. Such production, however, is problematic in terms of cost, productivity, safety, and the like.
In recent years, production of proteins for pharmaceutical applications has been attempted with the use of yeast, for the purpose of complementing the drawbacks described above. However, substantially no such attempts have been put to practical use. Regarding antibodies having complicated structures, in particular, there are examples of expression of Fab, a single stranded antibody (ScFv), or the like (Biosci. Biotechnol. Biochem., 64: 2138-2144, 2000). Productivity, however, is very low in terms of a full-length antibody (Proc. Natl. Acad. Sci. U.S.A., 85: 8678-8682, 1988). An example of antibody production with the use of yeast (Pichia pastoris) that produces a mammalian N-binding sugar chain was reported recently (Nature Biotechnology, 24: 210-215, 2006), although this report does not refer to the yield. Thus, high-level production of antibodies in yeast is difficult. Causes thereof are considered to be insufficient secretion ability of yeast, degradation caused by protease, or the like.
As a means for resolving such problems, a method involving the use of a protease deficient strain was proposed (Enzyme and Microbial Technology, 26: 671-677, 2000; Protein Expr. Purif., 20: 485-491, 2000, Biosci. Biotechnol. Biochem., 66: 628-631, 2002). The inventors have also developed a method of using a protease, which is a protease A (PEP4), protease B (PRB1), or yapsin (YPS1) gene deficient strain, to inhibit degradation of an antibody (WO 2003/091431). As a method for improving protein productivity via gene introduction, a method for improving ScFv productivity by allowing coexpression of parts of molecular chaperones that assist formation of 3-dimensional structures of proteins on the endoplasmic reticulum, such as BiP (KAR2)/PDI, was reported (Nat. Biotechnol. 16: 773-777, 1998), although this method merely produces a fragment of a single-stranded antibody.
Also, many inducible or constitutive promoters have been developed and used for producing foreign proteins. When genes encoding foreign proteins are allowed to express at high levels with the use of potent promoters in cells or when proteins that are less likely to fold are produced, however, aggregation occurs in the endoplasmic reticulum (ER) and resulting proteins may be sometimes accumulated in cells. Further, secretory proteins and membrane proteins are translated into proteins, incorporated into the endoplasmic reticulum immediately thereafter, subjected to a given modification, and then transferred to the Golgi apparatus. In such a case, unfolded proteins may be sometimes accumulated in the endoplasmic reticulum for some reason. This is referred to as “endoplasmic reticulum stress.” Examples of causes for such endoplasmic reticulum stress include disturbance of modification (e.g., addition of a sugar chain or disulfide bond) that occurs in the endoplasmic reticulum and deteriorated transportation from the endoplasmic reticulum. Mammalian cells have an “unfold protein response (UPR)” mechanism as a means for reacting against such endoplasmic reticulum stress. For example, proteins accumulated in the endoplasmic reticulum are protected by inhibition of transcription, acceleration of folding induced by molecular chaperones, degradation of denatured proteins, or cell death via apoptosis.
As genes that regulate UPR, genes of IRE1α-XBP1, PERK-eIF-2α, and ATF6 animal cells are known. In case of yeast, Ire1p-Hac1p is the only gene that is known as such gene, and the Ire1p-Hac1p gene is associated with UPR by the mechanism shown below (see FIG. 1). First of all, Ire1p is generally bound to an antibody heavy chain binding protein (BiP). When an unfolded protein (UFP) is produced, however, BiP binds to such UFP. Ire1p dissociated from BiP is activated via autophosphorylation or dimerization, and it exhibits endonuclease activity. While the HAC1 gene is generally in an inactivated state, Ire1p having endonuclease activity subjects mRNA transcribed from the HAC1 gene to splicing and produces active Hac1p (Cell, 87: 405-413, 1996; Cell, 90: 1031-1039, 1997; the EMBO Journal, 18: 3119-3132, 1999). Such active Hac1p migrates to the nucleus, acts as a transcription factor, and promotes expression of genes encoding various proteins associated with a series of reactions referred to as UPR, e.g., associated sugar chain addition, protein folding, protein degradation (ER-associated degradation: ERAD), protein sorting, lipid metabolism, or the like (Cell, 101: 249-258, 2000).
Regarding an attempt to improve productivity of foreign proteins utilizing activated Hac1p, there is an example in which the gene encoding activated Hac1p of a filamentous bacterium, i.e., Trichoderma reesei, is introduced into S. cerevisiae to improve secretion of a heterogeneous protein, α-amylase, and an endogenous protein, i.e., invertase, (Appl. Environ. Microbiol. 69: 2065-2072, 2003). However, a single protein, α-amylase or invertase, has been known as a protein that is easily secreted, and improvement in the amount of production is as low as approximately two times the amount of production prior to the improvement. In recent years, it has been reported that an antibody fragment, Fab, was produced using Pichia pastoris in a strain into which the activated HAC1 gene had been solely introduced (Biotechnology and Bioengineering, 94: 353-361). Productivity improvement via introduction of the activated HAC1 gene is as low as approximately 1.3 times.
Meanwhile, an example in which the mammalian-derived RRBP1 (ribosome-binding protein1, ribosome receptor, p180 protein) gene is solely introduced into a yeast strain, so as to quintuple the amount of proteins (bovine pancreatic trypsin inhibitor (BPTI)) secreted is known (The Journal of Cell Biology, 146: 273-284, 1999). At first, the RRBP1 gene was isolated from a dog as a gene encoding a protein binding to the ribosome (Nature, 346: 540-544, 1990). RRBP1 has a molecular weight of 180 kDa and a special structure such that a sequence comprising 10 amino acid residues on the N-terminal side is repeated 54 times and this region is bound to a ribosome. RRBP1 is known to be involved in enlargement of membrane structure and in stabilization of mRNA (the Journal of Cell Biology, 130: 29-39, 1995; the Journal of Cell Biology, 156: 993-1001, 2002). A successful example in the aforementioned BPTI has a molecular weight of 6,500, which represents a very small peptide. Such results cannot always be applied to other high-molecular-weight proteins or protein aggregates composed of different proteins such as light-chain or heavy-chain of antibodies.
The protein O-mannosyltransferase (PMT) gene is known to be associated with formation of O-sugar chains that are inherent to yeast or mold. The PMT gene product is localized on the ER membrane and has activity of adding mannose to a hydroxyl residue of serine (Ser) or threonine (Thr) of a secretory protein (hereafter, such activity is referred to as PMT activity). Some proteins to which sugar chains had been added by PMT serve as primary components of the yeast strain wall as mannoproteins. When PMT activity is extremely lowered, the cell wall is known to become fragile and to affect the growth of cells.
Regarding the PMT genes, the existence of seven genes, i.e., PMT1, 2, 3, 4, 5, 6, and 7, is known in Saccharomyces cerevisiae (S. cerevisiae) (Biochim. Biophys. Acta., 1426: 297-307, 1999). The PMT gene is classified into three types; i.e., the PMT1 family, the PMT2 family, and the PMT4 family. It is known that PMT1p and PMT2p exhibit activity upon formation of heterodimers, and PMT4p exhibits activity upon formation of homodimers. Because of amino acid sequence homology and the like, it is said that PMT5p complements PMT1p, and that PMT3p complements PMT2p. PMT6p is highly homologous to PMT2p and PMT3p, although the type of composite that exhibits activity is not known. Also, each PMT protein is known to have selectivity for a substrate protein.
As PMT genes of other types of budding yeast, five genes highly homologous to the PMT1, 2, 4, 5, and 6 genes of S. cerevisiae in the case of Candida albicans (Mol. Microbiol., 55: 546-560, 2005), a gene highly homologous at least to the PMT4 gene of S. cerevisiae in the case of Cryptococcus neoformans (Eukaryot. Cell, 6: 222-234, 2007), and three genes (oma1, 2, and 4) highly homologous to the PMT1, 2, and 4 genes in the case of fission yeast, i.e., Schizosaccharomyces pombe, (Mol. Microbiol., 57: 156-170, 2005) have been discovered. Further, the existence of five genes that are highly homologous to the PMT1, 2, 4, 5, and 6 genes of S. cerevisiae was observed in methanol-assimilating yeast, Ogataea minuta (O. minuta).
The PMT gene is also found in mold. The PmtA gene and two other genes are found in Aspergillus nidulans, and the Pmt1 gene, which is highly homologous to the PMT2 gene of S. cerevisiae, is found in Trichoderma reesei (Curr. Genet., 43: 11-16, 2003).
PMT activity is said to have effects of acting on a peptide hydrophobic region, enhancing peptide hydrophilicity, and inhibiting peptide aggregation in ER cavity. When foreign proteins are produced, however, PMT activity occasionally adds an unnecessary O-sugar chain, which may result in insufficient formation of protein composites, lowered activity, or the like. For multimeric proteins, such as antibodies, in particular, formation of aggregates thereof (which refers to formation of light chain and heavy chain aggregates, in the case of antibodies) may be inhibited.
JP Patent No. 3630424 and JP Patent Publication (kohyo) No. H08-509867 (A) (1996) propose a method for producing a recombinant protein via inhibition of O-sugar chain addition resulting from modification of the PMT gene. These patent documents, however, do not describe the formation of aggregates of a light-chain and a heavy-chain of antibody.
An example in which PMT1 and PMT2 gene-deficient strains associated with formation of O-sugar chains are used to inhibit addition of O-sugar chains to promote aggregation of antibody light-chain and heavy-chain molecules by approximately 1.5 times is provided in WO 2002/046437. This data is the result of a pulse-labeling experiment using an RI-labeled amino acid, but it is not the result of observing the entire culture process. Also, a degree of inhibition of sugar chain addition is further lowered, and antibody productivity is deteriorated.
Although the HAC1 gene induces UPR, some of the UPR-inducible genes are known to be PMT genes that add yeast-specific O-sugar chains (Cell, 101: 249-258, 2000). Accordingly, introduction of the HAC1 gene may not be sufficient to produce high-quality multimeric proteins, such as antibodies.
As described above, a variety of methods have been proposed as methods for high-level secretory production of proteins in yeast. However, substantially no method is sufficient at a practical level. A method for efficiently producing proteins, in particular, high-molecular-weight proteins or protein aggregates, including antibodies, has not yet been discovered. When a trait is introduced into a cell via gene introduction, gene destruction, or the like, in general, the cell would experience a given sort of stress. Thus, other modification may be provided, or an opposite action may occur. When high-level protein expression is intended, for example, UPR becomes activated, which results in a negative element, such as sugar chain modification, degradation by a proteasome, or ER-associated degradation (ERAD). Accordingly, high-level secretory production of proteins having complicated structures, such as antibodies, are not achieved by a single process, such as introduction of a single gene. Also, mere combination of several conventional methods would not always yield synergistic effects.
Accordingly, the present invention is intended to provide a method for high-level secretory production of proteins and, more particularly, proteins having complicated structures, such as antibodies, in yeast or other host cells.